JP2003052373A - Sensor tip using calcium ion dependent short chain deoxyribozyme and method for detecting base sequence - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a sensor tip using calcium dependent short chain deoxyribozyme and a method for detecting a base sequence having higher analytical accuracy than a conventional method by, for example, immobilization of a DNA or a RNA containing a specific base sequence on a DNA microarray and by detecting through two means comprising detection of a gene sequence (RNA) fragment formed by a catalytic reaction in the presence of calcium ion and detection (the conventional method) through a complementary binding in the absence of the calcium ion, noting that expression of a catalytic activity of a ribozyme having a specific sequence depends on the calcium ion. SOLUTION: A sensor tip binding a base sequence having a specific sequence structure (dGGCTACAACGA) on a surface of a base substance is constituted.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a sensor chip in which a calcium ion-dependent short-chain deoxyribozyme having a specific sequence structure is immobilized on a substrate for performing gene sequence analysis, and a nucleotide sequence detection method.

[0002]

In recent years, genes (DNA and R
Various detection methods have been developed for the purpose of analyzing the nucleotide sequence of (NA). One of the methods is the DNA microarray method. This method is to prepare a substrate on a substrate such as a slide glass in which a model DNA having a known base sequence is preliminarily solid-phase bound, and the model DNA on the DNA microarray is extracted from the inside of the cell and labeled with a fluorescent substance at the end. Liquid phase reaction.

When the base sequence of the model DNA and the base sequence of the test sample are complementary to each other, or when the substance in the test sample interacts with the model DNA,
It is known to identify a target substance by detecting fluorescence from a fluorescent substance that labels only a substance in a test sample that binds to model DNA.

However, in this DNA microarray method, the target gene in the model DNA or the test sample may form a higher-order structure, so that non-complementary / non-specific binding occurs and the accuracy of analysis is limited. was there. In order to reduce this non-complementary / non-specific binding, the washing operation to be carried out subsequently has been devised conventionally. For example, although measures have been taken by using a surfactant, adjusting the reaction temperature, and lengthening the washing operation, the washing operation is complicated and a sufficient reduction effect cannot be obtained. In fact, the gene in the test sample extracted from the living body has a larger number of bases (higher molecular weight) than the model DNA, and there is a possibility that the gene will bind even if the base sequence in the sample does not completely match the base sequence of the model DNA. high.

Further, in the DNA microarray method in which a large number of model DNAs having various base sequences are immobilized, each model DNA is
It was virtually impossible to set a reaction temperature suitable for the reaction, and it was virtually impossible to prevent non-complementary binding.

In recent years, deoxyribozymes and ribozymes (collectively referred to as ribozymes) having catalytic activity on DNA or RNA themselves have been found. It was revealed that this ribozyme requires a divalent metal ion such as calciur for catalytic activity. The inventors of the present application have discovered a specific nucleotide sequence (dGGCTACAACGA) that causes a catalytic activity function of site-specifically cleaving an RNA chain by using calcium ion, which is one of divalent metal ions, as an active factor.

The object of the present invention is to focus on the fact that the ribozyme catalytic activity consisting of this specific sequence depends on calcium ions. For example, DNA containing a specific base sequence is immobilized on a DNA microarray, and the presence of calcium ions in the presence of calcium ions. By using two methods, detection of a gene (RNA) fragment cleaved site-specifically by the catalysis of DNA and detection by complementary binding in the absence of calcium ions (conventional method), It is to provide a sensor chip and a base sequence detection method using a calcium ion-dependent short-chain deoxyribozyme with high analysis accuracy.

[0008]

The present invention is characterized in that a short-chain deoxyribozyme having a loop sequence having a specific sequence structure (dGGCTACAACGA) is bound to the surface of a substrate to form a sensor chip.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below with reference to the drawings. In FIGS. 1 and 2, the substrate 1 is composed of, for example, a slide glass, a column-packed bead, an SPR sensor chip, a crystal oscillator, or an electrode, and a ribozyme sensor chip 3 is fixedly bonded to the surface of the substrate 1 to form a ribozyme sensor chip 3. Constitute.

The base sequence of ribozyme is in the central part of the sequence.
It has a loop sequence of 11 nucleotides (dGGCTACAACGA),
It is composed of oligonucleotides designed so as to have complementary sequences to the nucleotide sequence to be detected at both ends of 11 nucleotides. The DNA used for this verification was subjected to solid-phase synthesis by the phosphoramidite method, followed by deprotection, and then only the DNA having the target chain length was separated and purified by gel electrophoresis or high performance liquid chromatography.

The above-mentioned ribozyme is solid-phase bonded to the surface of the substrate 1 by the following method to manufacture the ribozyme sensor chip 3.

(1). Biotinylated Immobilization Method Avidin (streptavidin) is immobilized on the surface of the substrate 1, and biotin is modified at the 5'end of the ribozyme. The biotin-modified ribozyme is spotted on the surface of the substrate 1 and the ribozyme is solid-phase bound by the specific binding of avidin and biotin.

(2). Thiolation immobilization method ≡. A gold thin film is vapor-deposited on the surface of the substrate 1, and the 5'end of the ribozyme is modified with thiol. The thiol-modified ribozyme is spotted on the surface of the substrate 1, and the ribozyme is solid-phase bound by the specific binding of the gold thin film and thiol.

≡. After aminating the surface of the substrate 1 with poly-L-lysine, aminopropylethoxysilane or the like,
SMCC (Succinimidyl 4 -‐ (N‐maleimidomethyl) Cyclohex
Ane-1-carboxylate) and the like are used for maleimidation and ribozyme 5 ′ end is modified with thiol. The thiol-modified ribozyme is spotted on the surface of the substrate 1 and solid-phase bonded by the specific bond between the amino group and the thiol group.

(3). Amination fixation method ≡. The surface of the substrate 1 is amino-grouped with poly-L-lysine, aminopropylethoxysilane or the like to be positively charged, and then a ribozyme is spotted, and a negative charge derived from the phosphate group of the ribozyme is electrostatically coupled to the positive charge of the amino group. By doing so, solid phase binding is achieved.

≡. The surface of the substrate 1 is amino-grouped with poly-L-lysine, aminopropylethoxysilane or the like to be positively charged and then ribozyme is spotted to electrostatically couple the negative charge derived from the phosphate group of the ribozyme to the positive charge of the amino group. Let Next, after drying by UV irradiation, blocking treatment with pyrrolidinone, succinic anhydride or the like is carried out, and solid phase binding is carried out.

≡. The surface of the substrate 1 is amino grouped with aminopropylethoxysilane or the like and then imidized with carbodiimide or the like, and a ribozyme or an amino-modified ribozyme whose 5'end is amidated is spotted on the surface of the imidized substrate 1 to form a solid phase. Join.

≡. After the surface of the substrate 1 is amino grouped with aminopropylethoxysilane or the like, PDC treatment is carried out, and P
A ribozyme or an amino-modified ribozyme whose 5'end is amidated is spotted on the surface of the DC-treated substrate 1 and solid-phase bound thereto.

≡. After the surface of the substrate 1 is converted to an aldehyde group with silylaldehyde or the like, a ribozyme or an amino-modified ribozyme whose 5'end is amidated is spotted on the surface of the aldehyde-modified substrate 1 and solid-phase bonded thereto.

≡. The surface of the substrate 1 is amino-modified with aminopropylethoxysilane and the like, and then an aldehyde is introduced to perform aldehyde formation, and then the substrate 1 is aldehyde-modified.
A ribozyme or an amino-modified ribozyme whose 5'end is amidated is spotted on the surface of the solid phase and bound to a solid phase.

The ribozyme bound to the surface of the substrate 1 in a solid phase as described above is, for example, cleaved, ligated or spliced.
It includes activities that catalyze chemical reactions such as out, splicing-in, and rearrangement.

Example of Verification of Catalytic Activity Ability in Calcium Ion Dependent Short Chain Deoxyribozyme As a substrate for a test sample, synthesis, deprotection and purification were performed in the same manner as ribozyme, and then fluorescent labeling was performed as shown in FIG. What was done was used. All RNA cleavage reactions were performed at 37 ° C in a buffer solution of 50 mM Tris_HCl (pH 8.0) adjusted to the desired concentration of divalent metal ions (Ca
²⁺ , Mg ²⁺ ) was added. The cleavage reaction was stopped by adding an aqueous solution of 100 mM Na ₂ EDTA and 9 M urea. Separation of substrate and cleavage products includes 7 M urea2
It was performed by gel electrophoresis using 0% denaturing polyacrylamide gel.

Quantification of the cleavage efficiency was performed using Fluor_S MultiImage
It was carried out by measuring the fluorescence intensity labeled with the 5'end of the substrate using r (BIO-LAD). Apparent reaction rate constant
The value of (k _obs ) was calculated by the curve fitting method using the following equation [1]. [P] = [P] SYMBOL 165 \ f "Symbol" \ s 12 SYMBOL 180 \ f "Symbol" \ s 12 (1− e ^{− kobs} SYMBOL 180 \ f "Symbol" \ s 12 ^t ) [1] [P ] Indicates the cleavage yield, [P] SYMBOL 165 \ f "Symbol" \ s 12 indicates the final cleavage yield, and t indicates the reaction time.

Short chain deoxyribozyme (dCGCTGGCA GGCTA
CAACGA GTCTTC, underline indicates loop site)
Substrate RNA in the presence of ²⁺ (rGAAGACA ↓ UGCCAGCG; ↓ indicates RNA cleavage site; U conventionally indicates uracil nucleotide)
Was cleaved. As a result, it was found that this short chain deoxyribozyme specifically cleaves the substrate RNA at one site between rAU. When the same RNA cleavage experiment was carried out in the absence of this short chain deoxyribozyme and in the absence of divalent metal ion, no RNA cleavage reaction was confirmed.

From these results, it was shown that the short-chain deoxyribozyme is a metal enzyme which requires a divalent metal ion as an active species for the RNA cleavage reaction. Also, as a result of calculating the apparent reaction rate constant from the measurement of the change in the cleavage yield during the reaction time, the value of k _obs at 37 ° C was 1.5 (SY
MBOL 177 \ f "Symbol" \ s 12 0.1) SYMBOL 180 \ f "Sym
bol "\ s 12 10 ^_1 min ^_1 .

In order to create a sensor chip using a short chain deoxyribozyme, ribozyme chip 3 immobilized on beads was used as a ribozyme sensor, and RNA cleavage reaction was carried out under the same conditions. As a result, the RNA cleavage site was specifically cleaved at one site between rAUs, as in the case without immobilization. Also, as a result of calculating the reaction rate constant,
The value of k _obs at 37 ℃ is 7.4 (SYMBOL 177 \ f "Symbol"
\ s 12 0.6) SYMBOL 180 \ f "Symbol" \ s 12 10 ^_2 min
It was shown to be ^_1 .

From these results, the ribozyme sensor comprising the ribozyme chip 3 has an apparent reaction rate slightly slower than that of the non-fixed ribozyme,
It was revealed that the target RNA was cleaved site-specifically.

Furthermore, although the ribozyme having this sequence did not show catalytic activity in the absence of calcium ions, hybridization formation with the complementary sequence was observed. In other words, it was confirmed that it does not show catalytic activity, but has the ability to discriminate complementary sequences. From this, it can be said that it is possible to select a two-step base sequence recognition means depending on the presence or absence of calcium ions under the catalytic reaction conditions.

Example of use of sensor chip The structure of the sensor chip may be any structure as long as it can construct reaction regions of two or more channels independent of each other.

Example 1 In an SPR sensor chip having two parallel reaction regions, one kind of ribozyme was immobilized on the first channel (region) and the second channel (region) by the thiolation immobilization method. These two channels are independent of each other, and the test sample to be subsequently reacted does not mix between the two channels. The test sample was sent to the first channel under a buffer solution containing 25 mM Ca ²⁺ , and the second channel was set to 10
A test sample as a buffer solution containing 0 mM monovalent metal ion was sent.

As a result, an increase in the sensorgram, which is considered to be complementary binding, was detected in both of them, but when the test sample solution was sent and then only the buffer solution was sent, the first channel containing calcium ions was detected. , A decrease in the sensorgram, which was suggested as a catalytic reaction, was observed, while in the second channel, an increase in the sensorgram, which seems to retain the complementary binding, was observed.

Example 2 Various types of solid phase-bound ribozymes are analyzed with a single sensor chip by using a DNA microarray.

The ribozyme and the nucleotide sequence nucleotides of only the fragment when catalytically decomposed by the ribozyme are used in the immobilization method (3). The DNA microarray sensor chip was prepared by immobilizing by the method described in the amination immobilization method i.

That is, FIG. 5 is an explanatory view showing a layout of ribozymes and fragment nucleotides to be immobilized, which shows a DNA microarray designed to the size of a slide glass.
The ribozyme and fragment nucleotides are immobilized on the left and right blocks (regions). The ribozyme and the fragment nucleotides immobilized on both blocks are the same.

Subsequently, a buffer solution containing 25 mM Ca ²⁺ was added as a fluorescent-labeled test sample to be reacted with the left block, and a buffer solution containing 100 mM monovalent metal ion was added to the right block, respectively, and reacted. Between the blocks, a rubber seal was attached between the blocks so that the test samples were not mixed with each other.

As a result, in the left block, fluorescence detection was not observed in the spot to which the ribozyme was immobilized, whereas in the right block, fluorescence from the spot to which the ribozyme was immobilized was observed. It was also shown that the detection intensity can be differentiated between the right side and the left side of the spot on which the fragment nucleotides are immobilized, and significant discrimination is possible.

[0037]

INDUSTRIAL APPLICABILITY The present invention focuses on the calcium ion dependence of the ribozyme catalytic activity consisting of a specific sequence.
DNA or RN containing a specific nucleotide sequence on A microarray
By immobilizing A and detecting the DNA fragment generated by the catalytic reaction in the presence of calcium ion, and detecting by complementary binding in the absence of calcium (conventional method), the conventional method can be used. It is possible to improve the analysis accuracy.

[Brief description of drawings]

FIG. 1 is a schematic part of a ribozyme chip.

FIG. 2 is a structural formula of a ribozyme bound to a solid phase.

FIG. 3 is a structural formula of a test sample.

FIG. 4 is an explanatory diagram showing a verification method.

FIG. 5 is an explanatory diagram showing a layout of ribozymes and fragment nucleotides to be immobilized.

[Explanation of symbols]

1-substrate, 3-ribozyme chip

[Procedure amendment]

[Submission date] August 2, 2002 (2002.8.2)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0013

[Correction method] Change

[Correction content]

(2). Thiolation immobilization method i. A gold thin film is vapor-deposited on the surface of the substrate 1, and the 5'end of the ribozyme is modified with thiol. The thiol-modified ribozyme is spotted on the surface of the substrate 1, and the ribozyme is solid-phase bound by the specific binding of the gold thin film and thiol.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0014

[Correction method] Change

[Correction content]

Ii. After aminating the surface of the substrate 1 with poly-L-lysine, aminopropylethoxysilane or the like,
SMCC (Succinimidyl 4 -‐ (N‐maleimidomethyl) Cyclohex
Ane-1-carboxylate) and the like are used for maleimidation and ribozyme 5 ′ end is modified with thiol. The thiol-modified ribozyme is spotted on the surface of the substrate 1 and solid-phase bonded by the specific bond between the amino group and the thiol group.

[Procedure 3]

[Document name to be amended] Statement

[Name of item to be corrected] 0015

[Correction method] Change

[Correction content]

(3). Amination fixation method i. The surface of the substrate 1 is amino-grouped with poly-L-lysine, aminopropylethoxysilane or the like to be positively charged, and then a ribozyme is spotted, and a negative charge derived from the phosphate group of the ribozyme is electrostatically coupled to the positive charge of the amino group. By doing so, solid phase binding is achieved.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0016

[Correction method] Change

[Correction content]

Ii. The surface of the substrate 1 is amino-grouped with poly-L-lysine, aminopropylethoxysilane or the like to be positively charged and then ribozyme is spotted to electrostatically couple the negative charge derived from the phosphate group of the ribozyme to the positive charge of the amino group. Let Next, after drying by UV irradiation, blocking treatment with pyrrolidinone, succinic anhydride or the like is carried out, and solid phase binding is carried out.

[Procedure Amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0017

[Correction method] Change

[Correction content]

Iii. The surface of the substrate 1 is amino grouped with aminopropylethoxysilane or the like and then imidized with carbodiimide or the like, and a ribozyme or an amino-modified ribozyme whose 5'end is amidated is spotted on the surface of the imidized substrate 1 to form a solid phase. Join.

[Procedure correction 6]

[Document name to be amended] Statement

[Correction target item name] 0018

[Correction method] Change

[Correction content]

Iv. After the surface of the substrate 1 is amino grouped with aminopropylethoxysilane or the like, PDC treatment is carried out, and P
A ribozyme or an amino-modified ribozyme whose 5'end is amidated is spotted on the surface of the DC-treated substrate 1 and solid-phase bound thereto.

[Procedure Amendment 7]

[Document name to be amended] Statement

[Correction target item name] 0019

[Correction method] Change

[Correction content]

V. After the surface of the substrate 1 is converted to an aldehyde group with silylaldehyde or the like, a ribozyme or an amino-modified ribozyme whose 5'end is amidated is spotted on the surface of the aldehyde-modified substrate 1 and solid-phase bonded thereto.

[Procedure Amendment 8]

[Document name to be amended] Statement

[Correction target item name] 0020

[Correction method] Change

[Correction content]

Vi. The surface of the substrate 1 is amino-modified with aminopropylethoxysilane and the like, and then an aldehyde is introduced to perform aldehyde formation, and then the substrate 1 is aldehyde-modified.
A ribozyme or an amino-modified ribozyme whose 5'end is amidated is spotted on the surface of the solid phase and bound to a solid phase.

[Procedure Amendment 9]

[Document name to be amended] Statement

[Name of item to be corrected] 0022

[Correction method] Change

[Correction content]

Example of Verification of Catalytic Activity Ability in Calcium Ion Dependent Short Chain Deoxyribozyme As a substrate for a test sample, synthesis, deprotection and purification were performed in the same manner as ribozyme, and then fluorescent labeling was performed as shown in FIG. What was done was used. All RNA cleavage reactions were performed at 37 ° C in a buffer solution prepared in 50 mM Tris-HCl (pH 8.0), and the divalent metal ion (Ca
²⁺ , Mg ²⁺ ) was added. The cleavage reaction was stopped by adding an aqueous solution of 100 mM Na ₂ EDTA and 9 M urea. Separation of substrate and cleavage products includes 7 M urea2
It was performed by gel electrophoresis using 0% denaturing polyacrylamide gel.

[Procedure Amendment 10]

[Document name to be amended] Statement

[Name of item to be corrected] 0023

[Correction method] Change

[Correction content]

Quantification of the cleavage efficiency was carried out by Fluor-S MultiImag.
er (BIO-LAD) was used to measure the fluorescence intensity at which the 5'end of the substrate was labeled. The value of the reaction rate constant (k _{o bs)} Apparent was calculated by the following [1] curve fitting method using the equation. [P] = [P] _∞ × (1−e ^{− kobs × t} ) [1] [P] represents the cleavage yield, [P] _∞ represents the final cleavage yield, and t represents the reaction time.

[Procedure Amendment 11]

[Document name to be amended] Statement

[Name of item to be corrected] 0025

[Correction method] Change

[Correction content]

From these results, it was shown that the short-chain deoxyribozyme is a metal enzyme which requires a divalent metal ion as an active species for the RNA cleavage reaction. Further, as a result of calculating the apparent reaction rate constant from the measurement of the reaction time change of the cleavage yield, the value of k _obs at 37 ° C was 1.5 (±
0.1) × 10 ^-1 min ^-1 .

[Procedure Amendment 12]

[Document name to be amended] Statement

[Correction target item name] 0026

[Correction method] Change

[Correction content]

In order to create a sensor chip using a short chain deoxyribozyme, ribozyme chip 3 immobilized on beads was used as a ribozyme sensor, and RNA cleavage reaction was carried out under the same conditions. As a result, the RNA cleavage site was specifically cleaved at one site between rAUs, as in the case without immobilization. Also, as a result of calculating the reaction rate constant,
The value of k _obs was shown to be 7.4 (± 0.6) × 10 ^-2 min ^{-1 at} 37 ℃.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/53 C12N 15/00 ZNAF 37/00 102 A F term (reference) 4B024 AA11 AA19 AA20 CA01 CA09 CA11 CA20 HA11 HA13 HA14 HA20 4B029 AA07 AA21 AA23 BB20 CC01 CC02 CC03 CC08 FA12 FA15 4B063 QA01 QA13 QQ42 QQ52 QR01 QR14 QR32 QR35 QR38 QR55 QR84 QS34 QS36 QS39 QX02 QX10

Claims (7)

[Claims] 1. A specific array structure (dGGCTACAACG) on the surface of a substrate.
A sensor chip on which a calcium ion-dependent short-chain deoxyribozyme having A) is immobilized. 2. A gene catalyzed by deoxyribozyme
The sensor chip using the calcium ion-dependent deoxyribozyme according to claim 1, wherein DNA or RNA having a base sequence complementary to the (RNA) fragment is bound. 3. By using a deoxyribozyme having a base sequence of a specific sequence structure (dGGCTACAACGA), by utilizing the catalytic reaction of deoxyribozyme caused by the presence or absence of a divalent metal ion, of
A detection method for detecting a DNA base sequence. 4. A test sample dropping area in a sensor chip is divided into at least two areas. One area is the area where the test sample containing calcium ions is dropped, and
The sensor chip using the calcium ion-dependent short-chain deoxyribozyme according to claim 1, wherein the other region is a region where a test sample containing no calcium ion is dropped. 5. A test sample dropping area in a sensor chip is divided into at least two areas. One area is the area where the test sample containing calcium ions is dropped, and
The sensor chip using the calcium ion-dependent short-chain deoxyribozyme according to claim 1, wherein the other region is a region where a test sample containing no magnesium ion is dropped. 6. A monovalent metal ion is added to a test sample containing no calcium ion or magnesium ion, and the mixture is added dropwise, and the binding with a ribozyme is compared with that of a test sample containing calcium ion or magnesium to enable analysis. A sensor chip using the calcium-dependent short-chain deoxyribozyme according to claim 3 or 4. 7. A short chain deoxyribozyme to be immobilized or
A sensor chip using the calcium ion-dependent short-chain deoxyribozyme according to claim 1, wherein DNA is bound at a desired density.